To make practical use of the complete sequence of the human and murine genomes, it is necessary to determine the biological function of individual genes. Selective gene inactivation (knockout) in the mouse in embryonic stem (ES) cells using gene trap vectors has emerged as a powerful experimental tool in this regard.
Most mammalian genes are divided into exons and introns. Exons are the portions of the gene that are spliced into mRNA and encode the protein product of a gene. In genomic DNA, these coding exons are divided by noncoding intron sequences. Although RNA polymerase transcribes both intron and exon sequences, the intron sequences must be removed from the transcript so that the resulting mRNA can be translated into protein. Accordingly, all mammalian, and most eukaryotic, cells have the machinery to splice exons into mRNA.
Gene trap vectors have been designed to integrate into genes in a manner that allows the cellular splicing machinery to splice vector encoded exons to cellular mRNAs. Commonly, gene trap vectors contain selectable marker sequences that are preceded by strong splice acceptor sequences and are not preceded by a promoter. Thus, when such vectors integrate into a gene, the cellular splicing machinery splices exons from the trapped gene onto the 5′ end of the selectable marker sequence. Typically, such selectable marker genes can only be expressed if the vector has integrated into a gene with an active promoter. The resulting gene trap events are subsequently identified by selecting for cells that can survive selective culture. Thus, not only does the insertion of the gene trap vector create a mutation in the trapped gene, it also provides a molecular tag for ease of identifying the gene that has been trapped. Common gene identification protocols used to obtain sequences from fusion transcripts include 5′ RACE, cDNA cloning, and cloning of genomic DNA surrounding the site of vector integration. However, these methods have proven labor intensive, not readily amenable to automation, and generally impractical for high-throughput. Moreover, such methods exclude the study of transcriptionally silent genes.
Other vectors have been developed that rely on a selectable marker gene preceded by a promoter and followed by a splice donor sequence instead of a polyadenylation sequence. However, these vectors do not result in expression of the selection marker unless they integrate into a gene and subsequently trap downstream exons which provide a polyadenylation sequence. Integration of such vectors into the chromosome results in the splicing of the selectable marker gene to 3′ exons of the trapped gene. These vectors do provide a number of advantages. They can be used to trap genes regardless of whether the genes are normally expressed in the cell type in which the vector has integrated. In addition, cells harboring such vectors can be selected and the trapped gene sequence can be identified using automated (e.g., 96-well plate format) gene identification assays such as 3′ RACE (see generally, Frohman, 1994). Using these vectors it is possible to produce large numbers of mutations and rapidly identify the mutated, or trapped, gene.
Although the use of ES cells in which genes have been trapped or inactivated is a powerful way to rapidly perform gene targeting for testing in a whole organism, e.g., a transgenic mouse (see Zahnbrowicz et al., 1998; Wiles et al., 2000), this method is limited because the gene is irreversibly inactivated in the ES cells. And although it is very useful to have the capability to knock out or inactivate a gene, the inactivation of many genes will be lethal or result in developmental adaptations. To individually target genes by homologous recombination and allow for conditional control of gene inactivation, recombinase/recognition site techniques, e.g., Cre-lox, have been used so that the resulting mice undergo site-specific recombination in a tissue-specific or temporally controlled manner. However, the production of mice with the conditional knockout is even more time-consuming and expensive than the production of traditional knockout mice. Moreover, the process of targeting requires extensive knowledge of the structure of a gene, a partial clone of the targeted gene, precise placement of recombinase recognition sites, and entails extensive manipulation of mouse genomic DNA.
Thus, what is needed is a high-throughput method to prepare a library of cells in which each cell in the library contains a gene that can be inactivated in a conditional, e.g., temporally- or spatially-controlled, manner. What is also needed is a rapid and efficient method to prepare targeting vectors for homologous recombination in a manner that results in a functional gene that can be disrupted in a conditional manner.